DESCRIPTION: On resting platelets, the integrin GPIIb-IIIa exists in a "resting" or inactive conformation, with a low affinity state. Platelets acquire the capacity to bind fibrinogen by a process of "inside-out" signaling, which results in the conversion of GPIIb-IIIa to an "active" conformation or high affinity state. The general aim of this proposal is to map signaling pathways leading to the activation of the platelet fibrinogen receptor, GPIIb-IIIa. A model is proposed for integrin activation in which step 1 involves an initial disruption of whatever interactions are required to maintain GPIIb-IIIa in a low affinity state, presumably by the addition or removal of platelet factor(s) from the cytoplasmic domain of GPIIb and/or GPIIIa. This is followed by step 2, which involves a mechanism to maintain GPIIb-IIIa in an activated state for a given period of time, presumably by interaction of platelet factor(s) with the GPIIIa cytoplasmic domain. Specific Aim 1 will further characterize the structure and function of a novel protein recently cloned and sequenced in this laboratory, termed CIB (Ca2+ and integrin binding protein) which is a candidate regulatory molecule for GPIIb-IIIa. Experiments are proposed to localize CIB in platelets and other cells by immunofluorescence microscopy, and to identify conditions in which CIB and GPIIb-IIIa may co-localize with one another. Further studies will identify sites on CIB and on the GPIIb cytoplasmic domain that bind to one another, identify other molecules that might bind to CIB, characterize posttranslational and cotranslational modifications of CIB (phosphorylation and/or myristoylation), and examine the effect of CIB on the ligand-binding function of purified GPIIb-IIIa. Specific Aim 2 will use CHO cells as a model cell line to dissect signal transduction pathways leading to step 1 (activation) and step 2 (maintenance of the activation state) of GPIIb-IIIa. To this end, experiments will be performed to determine the role of small G-proteins, (Ras and related proteins) and CIB in converting GPIIb-IIIa to an active fibrinogen receptor. Since it has been shown that R-Ras expression activates GPIIb-IIIa in CHO cells, R-Ras protein and other related molecules will be microinjected into CHO cells to determine whether they directly or indirectly (e.g., by inducing synthesis of other proteins) activate the integrin. Pharmacologic probes will be used to dissect the signaling pathway between R-Ras and integrin activation in CHO cells. Finally, attempts will be made to disrupt the activation of GPIIb-IIIa in CHO cells that express a constitutively active GPIIb-IIIa mutant in order to elucidate the signaling events required to maintain GPIIb-IIIa in its active state.